Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryote cells. They consist of six different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested in not only the structure, function and expression of keratin IF of human skin and their roles in keratinopathy diseases, but also of the related IF of other cell types in order to understand their roles in biology. Ongoing structural studies While the roles of the keratins in many genetic diseases are now well understood, further structural studies are necessary to develop rational approaches to therapy. We have initiated a major study in collaboration with other investigators in Switzerland, Germany and New Zealand to solve the three-dimensional structure of vimentin IF by use of Xray crystallographic techniques. These IF have been chosen because: (a) they are homopolymeric, and therefore likely to be somewhat simpler to solve; and (b) they have a very high sequence homology with keratin IF, and thus many of the structural principles used for vimentin should be applicable to keratin IF. To date, we have solved the structure of the last 35 residues of the 2B rod domain segment, which encompasses the helix-termination motif, and the likely trigger motif required for successful IF assembly. Work is now in progress on numerous other constructs have been made which cumulatively cover the entire rod domain portion of vimentin. The role of ionic interactions in IF structure: the presence of trigger motifs Current evidence suggests that the packing of protein molecules within IF is in part governed by favorable ionic interactions between charged residues along the rod domain segments of the IF chains. Using the type I/II keratin 5/14 paradigm, we have identified two regions along the chains that are absolutely required for the formation of a two-chained coiled-coil molecule. These are residues 100-113 of the 2B rod domain segment, and residues 79-93 of the 1B rod domain segment. These probably form triggers which are necessary to stabilize and/or initiate coiling of the two chains to form a coiled-coil molecule. Also residues Glu106 and Lys23 of the 2B rod domain segment are required to specify and stabilize the A22 mode of alignment of a pair of molecules. Likewise, residue 10 of the 1A rod domain segment, and residues 4-7 of the L2 linker are required to specify and stabilize the A11 alignment mode of two molecules. These studies have now defined key residues required for the first two hierarchical levels of keratin IF structure. Parallel studies are now in progress on the type III vimentin system: preliminary data documents that different residues are in fact required for maintenance of the A11 and A22 molecular alignments. The organization of molecules in trichocyte keratin IF We have previously demonstrated by detailed cross-linking experiments that pairs of epidermal keratin molecules are aligned in three modes termed A11, A22 and A12. When assimilated into IF, pairs of molecules in the same axial row adopt a fourth mode termed ACN, in which the end of one molecule overlaps the beginning of the adjacent molecule by about 1 nm. Interestingly, almost all known keratinopathy mutations/substitutions reside in this overlap window. We also have shown that the molecules of type III IF adopt the same basic four modes, but the alignments of the former three are slightly offset. This adequately explains why type III and types I/II keratin chains cannot and do not coassemble in vivo or in vitro. We have now re-examined these questions in trichocyte IF, and have found that the alignments for reduced trichocyte keratin IF are exactly the same as for cytokeratin IF. However, when trichocyte keratins are oxidized, a series of disulfide bonds form that shift the A11 alignment mode, with the net result of a 1 nm gap between the end and beginning of adjacent molecules. Interestingly, several of the disulfide bonds occurred between head domain and rod domain cysteines, which suggests that the head domain is intimately involved in the molecular shifts and is essential for the maintenance of stability of the trichocyte IF structure. The only way this could occur is if the head domain sequences fold back over the rod domain. This may explain why head domain sequences are essential for successful IF assembly. Tests of this hypothesis will be extremely difficult for trichocyte IF, however, due to the probability of random disulfide interchange. Therefore, we plan to explore this issue with both type III vimentin and types I/II K5/K14 keratin filaments. Initially, we will use deletion cloning experiments coupled with more cross-linking studies to define precisely (a) which residues are essential for successful IF assembly; (b) which residues if any interact with which residues of the 1A and 1B rod domain segments; and (c) how. Structure of the end domains We have discovered a novel mutation in the gene encoding keratin 1 which causes a frameshift and change in the coding sequence of the tail domain. In the wildtype protein, such sequences are enriched in glycines, but in the mutant, the frame has been changed to alanine-rich instead. The net result is a severe case of ichthyosis hystrix Curth-Macklin. It appears the abnormal keratin tail allows IF to form, but severely interferes with keratin IF function. In particular, the distribution of loricrin to the cell periphery is prevented, resulting in a severe barrier function defect. This is not only the first mutation reported for a keratin end domain in disease, but now allows for new insights into the structure and function of the tail domains. Searches for high molecular weight IF associated proteins When type III vimentin IF are passaged through cycles of assembly and disassembly in vitro, certain high molecular weight proteins always co-cycle. We have purified one of these from BHK-21 fibroblasts, and shown that it is the type VI IF protein nestin. This protein can be incorporated in small amounts into vimentin IF and its long tail is thought to serve as an interfilamentous spacer to separate individual IF and affect supramolecular organization in cells. We believe that functionally similar molecules should exist for keratin IF. Keratin IF in epithelia usually occur in tonofibril bundles containing 100s of individual IF loosely separated by 1-3 filament diameters from each other. Ongoing experiments are designed to look for keratin IF organizing or spacer proteins in epithelial cells.